A type of swelling seahorse oil and its application in the preparation of anti-sarcopenia products

Abstract

This invention relates to the field of biotechnology, and more particularly to a swelling-promoting seahorse oil and its application in the preparation of anti-sarcopenia products. Obtained using a supercritical carbon dioxide extraction process, the swelling-promoting seahorse oil of this invention has been experimentally verified to inhibit muscle degradation, promote muscle cell activation and differentiation, increase muscle mass, and enhance muscle strength and endurance. Furthermore, it exerts its effects through dietary supplementation with no significant adverse reactions, and can be used to prepare anti-sarcopenia related foods, health products, or pharmaceuticals to meet the muscle health maintenance needs of the elderly.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a bloated seahorse oil and its application in the preparation of anti-sarcopenia products. Background Technology

[0002] Sarcopenia, an age-related geriatric syndrome, is characterized by decreased muscle mass and strength, with a particularly high incidence in people over 60 years of age. It significantly increases the risk of falls, fractures, and metabolic disorders, placing a heavy burden on the healthcare system. Currently, there are no effective treatments available clinically, dietary interventions have limited effectiveness, and hormonal drugs have significant adverse reactions. Therefore, there is an urgent need to develop safe and effective natural active products.

[0003] Seahorses, as a traditional medicinal marine organism, have lipid extracts rich in unsaturated fatty acids (such as DHA and arachidonic acid), high-quality protein, and various essential amino acids, which have been proven to possess anti-aging and antioxidant bioactivities. The swollen-bellied seahorse, a superior breed for large-scale aquaculture in my country, boasts advantages such as large size, strong disease resistance, and high survival rate. Its artificial breeding technology has achieved breakthroughs, ensuring the sustainable utilization of resources. Currently, seahorses are only used as one ingredient in traditional Chinese medicine prescriptions. For example, Chinese patent CN115300569A, "A Traditional Chinese Medicine Composition and Application for Treating Rheumatic Bone Pain and Muscle Injury," discloses a traditional Chinese medicine composition for treating rheumatic bone pain and muscle injury, comprising the following raw materials in parts by weight: borneol 2-6 parts, camphor 5-10 parts, myrrh 5-10 parts, safflower 2-10 parts, drynaria fortunei 6-10 parts, angelica pubescens 5-10 parts, dipsacus asper 5-10 parts, and achyranthes bidentata 5-10 parts. 0 parts, Rehmannia glutinosa 5-10 parts, Pinellia ternata 5-10 parts, Dragon's blood 5-10 parts, Angelica dahurica 5-10 parts, Ligusticum chuanxiong 5-10 parts, Asarum sieboldii 5-10 parts, Angelica dahurica 5-10 parts, Arisaema heterophyllum 5-10 parts, Vegetable oil 6-10 parts, Seahorse 2-10 parts, Seahorse 2-10 parts, Cynanchum paniculatum 5-10 parts, Cinnamomum cassia 5-10 parts, Sophora tonkinensis 5-10 parts, Atractylodes macrocephala 5-10 parts, Litharge 5-10 parts, Aconitum carmichaelii 2-10 parts, Aconitum kusnezoffii 2-10 parts, Phellodendron chinense 2-10 parts. The effects of seahorse are described as: sweet, salty, and warm; its functions and indications are: dispersing nodules and reducing swelling, treating swelling of the waist and legs, carbuncles and boils. However, the specific functional components of the hippocampus, such as its anti-sarcopenic activity, have not been identified. The application value of the hippocampus in the field of muscle protection has not yet been explored, and the relevant technological gaps provide a research foundation and innovation space for this invention. Summary of the Invention

[0004] The technical problem to be solved by this invention is that existing research has not explored the anti-sarcemic activity of expanded seahorse oil, and its application value in the field of muscle protection has not yet been discovered. Further research is needed to provide new raw materials for anti-sarcemic products.

[0005] To address the aforementioned problems, this invention provides a swelling seahorse oil and its application in the preparation of anti-sarcopenia products. The oil is obtained using a supercritical carbon dioxide extraction process. Experiments have verified that the swelling seahorse oil of this invention can inhibit muscle degradation, promote muscle cell activation and differentiation, increase muscle weight, and increase muscle strength and endurance, with effects superior to swelling seahorse powder.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] A method for preparing bloated seahorse oil includes the following steps: taking an appropriate amount of frozen bloated seahorse sample, removing impurities, washing and draining; drying with a freeze dryer, pulverizing and sieving to increase the specific surface area; loading the powder into an extraction vessel, starting the supercritical extraction system, pressurizing CO2 to 38 MPa via a high-pressure pump, and heating it to 42°C via a heater to reach a supercritical state; continuously feeding supercritical CO2 into the extraction vessel for extraction; the extracted CO2-oil mixture enters a separation vessel, where the CO2 loses its supercriticality by depressurization and cooling; the CO2 is vaporized and recycled, while the oil is collected at the bottom of the separation vessel to obtain refined bloated seahorse oil.

[0008] Furthermore, when supercritical CO2 is continuously introduced into the extraction vessel, the flow rate is controlled at 15 L / h, and dynamic extraction is performed for 3 hours. At the same time, 8% anhydrous ethanol is added as an entrainer to enhance the extraction efficiency of polar lipids.

[0009] A type of bloated seahorse oil prepared by the above method.

[0010] The above-mentioned application of swelling seahorse oil in the preparation of anti-sarcopenia products. Dietary swelling seahorse oil can significantly improve sarcopenia by inhibiting muscle degradation, promoting muscle cell activation and differentiation, increasing muscle mass, muscle strength and endurance. A 0.5% intake dose can produce significant effects, and is superior to 1% seahorse powder.

[0011] Further, cholesterol sulfate was extracted from the above-mentioned expanded seahorse oil. The specific steps are as follows: Take an appropriate amount of the above-mentioned expanded seahorse oil, dissolve it thoroughly in chloroform, and mix it with activated (105℃ for 12h) 200-300 mesh silica gel powder. Use a rotary evaporator (RE-2000A, Shanghai Yarong Biochemical Instrument Factory) to remove the chloroform. The specific conditions for rotary evaporation are: rotation speed 30-50 rpm / min, water bath temperature 45-50℃, and vacuum degree 0.09 MPa, so that the lipids are uniformly adsorbed in the silica gel powder. Add the dried silica gel powder containing total seahorse oil lipids to a silica gel column, and separate and purify the lipid components of seahorse oil by silica gel column chromatography. Elute with chloroform, chloroform:methanol (9:1, v / v), and chloroform:methanol (5:1, v / v), respectively. Detection is performed by thin-layer chromatography (TLC), with sulfate colorimetric analysis. Combine the eluents of the same components and remove the solvent using a rotary evaporator to obtain a mixture of seahorse oil steroids. Dissolve the above seahorse oil steroid mixture in a small amount of chloroform and uniformly load it onto the top of a new silica gel column. The elution gradient is as follows: petroleum ether: ethyl acetate = 9:1, flow rate 1-2 mL / min, to remove nonpolar impurities; petroleum ether: ethyl acetate = 7:3, flow rate 1-2 mL / min, to remove cholesterol; chloroform: methanol = 1:1, flow rate 1 mL / min, to elute cholesterol sulfate.

[0012] The above-mentioned cholesterol sulfate esters are used in the preparation of anti-sarcopenia products.

[0013] The beneficial effects of this invention are as follows:

[0014] (1) Supercritical carbon dioxide extraction process is used to efficiently extract fat from the bloated seahorse. The product is rich in active ingredients such as DHA+EPA (18.06%) and phospholipids. It has high purity and no solvent residue. The raw material is bloated seahorse raised on a large scale. The resources are sustainable. It is easier to absorb than seahorse powder and has greater application potential.

[0015] (2) Experiments have shown that the expanded seahorse oil of the present invention can inhibit muscle degradation, promote muscle cell activation and differentiation, increase muscle weight, increase muscle strength and endurance, and the effect is better than that of expanded seahorse powder; and it can play a role through dietary supplementation without obvious adverse reactions. It can be used to prepare anti-sarcopenia related foods, health products or medicines to meet the muscle health maintenance needs of the elderly.

[0016] (3) Cholesterol sulfate was further isolated from the bloated seahorse oil and confirmed that it can promote the proliferation of mouse myoblasts C2C12. At a concentration of 10 μM, the proliferation was increased by nearly 50%, providing a key material basis for anti-sarcopenia. Attached Figure Description

[0017] Figure 1The results of the mouse behavioral tests are shown below. Note: Different letters indicate that there are significant differences between different groups at the p < 0.05 level, as determined by analysis of variance (Tukey test). A represents the magnitude of the pulling force in the tensile test; B represents the suspension time in the suspension test.

[0018] Figure 2 The values ​​represent the mRNA expression levels of genes related to muscle degradation and differentiation in mice. Note: Different letters indicate significant differences between different groups at the p < 0.05 level, as determined by analysis of variance (Tukey test). Specifically, A represents the mRNA expression level of muscle ring finger protein 1 (MuRF1); B represents the mRNA expression level of muscle atrophy F-box protein (MAFbx); C represents the mRNA expression level of muscle differentiation factor MyoD; and D represents the mRNA expression level of myopoietin MyoG.

[0019] Figure 3 This indicates the detection of cell proliferation activity of hippocampal cholesterol sulfate using the CCK-8 assay. Note: Different letters indicate significant differences between different groups at the p < 0.05 level, as determined by analysis of variance (Tukey test). Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0022] Example 1:

[0023] A type of bloated seahorse oil is prepared by the following method:

[0024] A suitable amount of frozen, bloated seahorse samples (purchased from Weihai Yinze Biotechnology Co., Ltd.) were taken, impurities were removed, and the samples were washed and drained. Vacuum freeze-drying was performed using a freeze dryer (Alpha 1-4 LSC, Marin Christ GmbH, Germany) under the following conditions: temperature -50 ℃, vacuum degree < 10 kPa, and time 48 h. After freeze-drying, the samples were pulverized and passed through a 30-mesh sieve to increase the specific surface area. The powder was then loaded into an extraction vessel, and the supercritical extraction system was started. CO2 was pressurized to 38 MPa by a high-pressure pump and heated to 42 ℃ by a heater to reach a supercritical state. Supercritical CO2 was continuously introduced into the extraction vessel at a flow rate controlled at 15 L / h for dynamic extraction for 3 hours. Simultaneously, 8% anhydrous ethanol was added as an entrainer to enhance the extraction efficiency of polar lipids. The extracted CO2-oil mixture was then introduced into a separation vessel, where the CO2 lost its supercriticality by reducing the pressure (5-10 MPa) and temperature (25-30 ℃). The CO2 is vaporized and recycled, while the oil is collected at the bottom of the separator to obtain refined bloated seahorse oil. This oil is transferred to a liquid chromatography bottle, filled with nitrogen, sealed with a sealing film, and stored at -20°C for later use.

[0025] Result verification:

[0026] The fatty acid content of the above-mentioned expanded seahorse oil was determined by gas chromatography. The results showed that the total lipid content of the expanded seahorse oil was as high as 18.06% (DHA+EPA). Thin-layer chromatography results showed that the total lipid content of the expanded seahorse oil mainly consisted of free fatty acids, phospholipids, and triglycerides, and also contained a certain amount of steroidal compounds. The reagent kit (Nanjing Jiancheng) showed that the contents of free fatty acids and triglycerides in the total lipid content of the expanded seahorse oil were 33.5 mg / g dry weight and 6.8 mg / g dry weight, respectively. The phospholipid content in the total lipid content of the expanded seahorse oil was determined to be 12.5 mg / g dry weight by inorganic phosphorus detection method. Previous studies have shown that phospholipids and free fatty acids, especially EPA and DHA, promote muscle synthesis and repair by regulating inflammatory responses and the mTOR signaling pathway. Triglycerides, as an energy source, can provide sufficient energy for muscles to support their normal metabolism and growth. The above information suggests that the expanded seahorse oil of the present invention may have the potential function of improving sarcopenia.

[0027] Animal experiments verified the effect of distended seahorse oil on improving sarcopenia in mice:

[0028] Male 4-month-old SAMP8 rapidly aging mice (SPF grade, weight 22.0 ± 0.5 g) were purchased from Nanjing Qingzilan Technology Co., Ltd. Animal housing conditions were maintained at 21℃~23℃, humidity at 45%~55%, with a 12-hour light / 12-hour dark cycle and free access to water. Mice were first fed a high-fat diet for two consecutive months, then randomly divided into 5 groups: SAMP8 group (model group), SAMP8 + 0.2% bloated seahorse oil group (low-dose group), SAMP8 + 0.5% bloated seahorse oil group (medium-dose group), SAMP8 + 1% bloated seahorse oil group (high-dose group), and SAMP8 + 1% bloated seahorse powder group (positive control group), with 8 mice in each group. Bloated seahorse oil was administered for 3 consecutive months. One week before the start of behavioral studies, 2-month-old young SAMP8 mice were purchased as a normal control group and fed stick-shaped feed until culling for material collection.

[0029] Table 1 shows that, compared with the normal control group, the muscle index of the model group mice was significantly decreased, exhibiting characteristics of sarcopenia. After dietary supplementation with seahorse oil, the muscle weight and muscle index of mice in all groups significantly recovered, and were directly proportional to the intake dose. Notably, the effect of 0.5% seahorse oil was comparable to that of 1% seahorse powder.

[0030] Table 1 Comparison of total gastrocnemius muscle weight, body weight, and gastrocnemius muscle index among the groups of mice:

[0031] .

[0032] Forelimb tension results showed ( Figure 1 In the model group (A), compared with the normal control group, the forelimb muscle strength of mice was significantly reduced. Dietary supplementation with expanded seahorse oil significantly increased the forelimb muscle strength of mice in all groups, and this increase was directly proportional to the intake dose. Specifically, 0.2% expanded seahorse oil showed no significant improvement, while 0.5% and 1% expanded seahorse powder had comparable effects. 1% expanded seahorse oil showed the most significant effect in increasing forelimb muscle strength, restoring it to near-normal levels.

[0033] The results of limb suspension showed ( Figure 1 (B) 0.2% seahorse oil for swelling did not show any effect. 0.5% and 1% seahorse oil for swelling had comparable effects on improving limb muscle strength and were better than 1% seahorse powder for swelling.

[0034] Furthermore, the results of the mechanism investigation indicate that ( Figure 2In the study, dietary intake of 0.5% and 1% expanded seahorse oil significantly reduced the expression of muscle degradation-related genes MuRF1 (muscle ring finger protein 1) and MAFbx (muscle atrophy F-box protein), with better effects than 1% expanded seahorse powder. However, 0.2% expanded seahorse oil did not exert an inhibitory effect on muscle degradation due to its low intake dose. These results indicate that expanded seahorse oil can inhibit muscle degradation in a dose-dependent manner.

[0035] In addition, dietary bulking seahorse oil also promotes the activation and differentiation of muscle cells. Figure 2 Specifically, the expression levels of myoD (satellite cell activation marker) mRNA increased by 25% (p<0.05), 47.5% (p<0.05), and 100% (p<0.01) in the 0.2%, 0.5%, and 1% expanded seahorse oil groups, respectively, while the expression level of myopoietin (satellite cell differentiation marker) mRNA increased by 56.5% (p<0.01) in the 1% expanded seahorse oil group. This indicates that expanded seahorse oil has the most significant promoting effect on muscle cell differentiation at the 1% dose, and its overall effect is better than that of 1% seahorse powder.

[0036] Example 2: Study on the effect of expanded seahorse oil cholesterol sulfate on the proliferation of mouse C2C12 myoblasts:

[0037] Cholesterol sulfate was extracted and separated from total seahorse oil lipids using silica gel column chromatography. The specific steps are as follows: An appropriate amount of total seahorse oil lipids prepared in Example 1 was fully dissolved in chloroform and then mixed with activated (105℃ for 12h) 200-300 mesh silica gel powder. The chloroform was removed using a rotary evaporator (RE-2000A, Shanghai Yarong Biochemical Instrument Factory). The specific conditions for rotary evaporation were: rotation speed 30-50 rpm / min, water bath temperature 45-50℃, and vacuum degree 0.09 MPa, ensuring uniform adsorption of lipids onto the silica gel powder. The dried silica gel powder containing total seahorse oil lipids was added to a silica gel column, and the lipid components of seahorse oil were separated and purified using silica gel column chromatography. Elution was performed with chloroform, chloroform:methanol (9:1, v / v), and chloroform:methanol (5:1, v / v), respectively. Thin-layer chromatography (TLC) was used for monitoring and detection, with sulfate colorimetric analysis. Combine the eluents of the same components and remove the solvent using a rotary evaporator to obtain a mixture of seahorse oil steroids. Dissolve the above seahorse oil steroid mixture in a small amount of chloroform and uniformly load it onto the top of a new silica gel column. The elution gradient is as follows: petroleum ether: ethyl acetate = 9:1, flow rate 1-2 mL / min, to remove nonpolar impurities; petroleum ether: ethyl acetate = 7:3, flow rate 1-2 mL / min, to remove cholesterol; chloroform: methanol = 1:1, flow rate 1 mL / min, to elute cholesterol sulfate.

[0038] Cell proliferation assay: Mouse C2C12 myoblasts were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged after reaching the logarithmic growth phase. The effect of expanded hippocampal oil cholesterol sulfate on the cell proliferation capacity of mouse C2C12 myoblasts was detected using the CCK-8 colorimetric assay. After passage, C2C12 cells were dissolved in DMEM high-glucose medium containing 10% fetal bovine serum and seeded at 1×10^4 cells per well in 96-well plates. The cells were incubated at 37°C for 24 hours in a 5% CO2 incubator. Afterward, the supernatant was discarded from each well, and 100 μL of different concentration gradients (1 μM, 5 μM, 10 μM) of expanded hippocampal oil cholesterol sulfate prepared in DMEM high-glucose medium containing 10% fetal bovine serum were added to each well (five replicates per group). The cells were then incubated at 37°C for 24 hours in a 5% CO2 incubator. Finally, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 4 hours. The OD value of each well was measured at 450 nm, and the average OD value of all replicates was taken. Cell proliferation capacity % = (OD of sampled cells - OD of blank cells) / (OD of control cells - OD of blank cells) × 100%.

[0039] The results of the CCK-8 cell proliferation experiment showed that ( Figure 3 Hippocampus oil cholesterol sulfate significantly promoted the proliferation of mouse C2C12 cells in a dose-dependent manner. Compared with the normal control group, 1 μM cholesterol sulfate significantly increased the proliferation of C2C12 cells by 15.1% (p < 0.05); while the 5 μM and 10 μM concentrations showed the most significant effects on the proliferation of C2C12 cells, increasing it by 38.5% (p < 0.01) and 49.2% (p < 0.01), respectively, but there was no significant difference between the two concentrations.

[0040] In conclusion, dietary seahorse oil can inhibit muscle degradation, promote muscle cell activation and differentiation, increase muscle mass, and enhance muscle strength and endurance, with effects superior to those of seahorse powder. In particular, the cholesterol sulfate in seahorse oil can significantly enhance the proliferative capacity of myoblasts (C2C12), thereby improving sarcopenia.

[0041] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A method for extracting cholesterol sulfate from expanded seahorse oil, characterized in that... Includes the following steps: (1) Take an appropriate amount of frozen bloated seahorse sample, remove impurities, wash and drain; dry with a freeze dryer, pulverize and sieve; load the powder into the extraction vessel, start the supercritical extraction system, pressurize CO2 through a high-pressure pump, heat it through a heater to reach the supercritical state; continuously feed supercritical CO2 into the extraction vessel for extraction; the extracted CO2-oil mixture enters the separation vessel, and CO2 loses its supercriticality by depressurization and cooling; CO2 is recycled after gasification, and the oil is collected at the bottom of the separation vessel to obtain refined bloated seahorse oil; (2) Take refined bloated seahorse oil, dissolve it thoroughly in chloroform, mix it with activated silica gel powder, and remove the chloroform using a rotary evaporator; add the dried silica gel powder containing total seahorse oil lipids to a silica gel column, and separate and purify the lipid components of seahorse oil by silica gel column chromatography; elute with chloroform, chloroform:methanol (9:1 volume ratio), and chloroform:methanol (5:1 volume ratio), and monitor the eluents with thin-layer chromatography and perform sulfate colorimetric analysis; combine the eluents with the same components, remove the solvent using a rotary evaporator, and obtain the seahorse oil steroid mixture; dissolve the above seahorse oil steroid mixture in a small amount of chloroform, load it evenly onto the top of a new silica gel column, and elute to obtain cholesterol sulfate.

2. The method as described in claim 1, characterized in that: In step (1), when supercritical CO2 is continuously introduced into the extraction vessel, the flow rate is controlled at 15 L / h, and dynamic extraction is performed for 3 hours. At the same time, 8% anhydrous ethanol is added as an entrainer.

3. The method as described in claim 1, characterized in that: In step (2), the silica powder is 200-300 mesh silica powder activated at 105℃ for 12h.

4. The method as described in claim 1, characterized in that: The specific conditions for rotary evaporation in step (2) are a rotation speed of 30-50 rpm / min, a water bath temperature of 45-50℃, and a vacuum degree of 0.09 MPa.

5. The method as described in claim 1, characterized in that: The elution gradient in step (2) is as follows: petroleum ether: ethyl acetate ratio of 9:1, flow rate of 1-2 mL / min, to remove nonpolar impurities; petroleum ether: ethyl acetate ratio of 7:3, flow rate of 1-2 mL / min, to remove cholesterol; Chloroform:methanol ratio 1:1, flow rate 1 mL / min.

6. The use of the cholesterol sulfate prepared by any one of claims 1-5 in the preparation of anti-sarcopenia products.

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